Immunohistochemical (IHC) assays and assay techniques based on in situ hybridization (ISH) and, in particular, fluorescent in situ hybridization (FISH) are commonplace techniques used in medical diagnostics today. Biological samples such as tissue or cell samples from suspected or known diseased patients are analyzed using IHC and ISH/FISH techniques to determine or monitor the patient's status with respect to the disease under investigation. However, both IHC and ISH/FISH methods require the performance of numerous complex and time consuming steps in preparing the tissue and cell samples for and then carrying out the actual IHC or hybridization assay. When performed manually, these assay methods are tedious, technically demanding and time consuming. Nevertheless, well-established protocols have been established for manual assays.
As demand for both IHC and ISH/FISH assays has increased so also has the demand for automating these techniques to promote greater throughput, enhance reliability and spare technicians from the tedium and complexity of performing the assays. One artifact resulting from automation is the preferred use of smaller and more controlled amounts of sample and reagents in assay steps. For example, when FISH assays are performed manually sample washing, aging and pretreatment are typically performed using reagents in substantial excess such as dipping sample slides into Coplin jars filled with appropriate reagents. Much smaller reagent volumes (e.g., less than 1 ml) are highly desired for automated assays. Instrument designers are also keen to simplify assays for automated or semi-automated instrumentation.
FISH samples are typically treated in a buffered solution to accommodate digestion and/or washing. Such buffers typically include well-known buffers such as SSC (0.3M sodium chloride and 0.03M sodium citrate) or PBS (phosphate-buffered saline) at various concentrations. 2×SSC is typically used as a buffered post-hybridization wash reagent for formalin-fixed paraffin embedded (FFPE) tissue slides, and as a cell aging reagent for urine and amniotic fluid cell slides. Similarly, 0.4×SSC is typically used as a post-hybridization stringency wash for cell-based specimens. Wash reagents normally include a surfactant or detergent such as Nonidet P-40 (NP-40) to facilitate reagent spreading across the sample surface. Some investigators have found incubation of biological samples in high concentrations of glycerol in SSC (hot 2×SSC/50% glycerol) improves penetration of FISH probes in sample cells.
In an automated system, heating can be accomplished by placing a slide carrying the biological sample on the surface of a heating element. The biological samples are treated with reagents while the slide remains on the heating element. Assay reagents are usually applied mechanically. Many assay steps are performed at elevated temperatures. For example, in the Vysis PathVysion® HER-2 DNA Probe test breast tissue specimens are washed under stringent conditions following hybridization in a 2×SSC/NP-40 wash buffer at 73° C. for about 2 min or longer. However, assay steps at elevated temperatures can cause substantial evaporation of assay reagents. When small volumes are used, sample wash and aging steps and the resulting assay can be compromised by evaporation. Reagent evaporation has not been a concern in manual assays because sample slides are frequently covered during assay steps to prevent excess evaporation during the steps and Coplin jars or basins filled with excess reagents are used. For example, during the Vysis PathVysion® test, the cover slip is sealed to the sample slide using rubber cement to facilitate in situ hybridization. Following hybridization, the sealant is removed and the sample slide is immersed in wash buffer. Similarly, U.S. Pat. No. 6,855,559 to Christensen and U.S. Pat. No. 6,855,552 to Towne, et al. teach the use of the water immiscible Liquid Coverslip™ sold by Ventana Medical Systems to inhibit evaporation during automated IHC and ISH assays.
Designers of automated assays place a premium on the use of much smaller and more carefully controlled reagent volumes than are used in manual assays. Similarly, designers place a high premium on the ability to perform assay steps in an open and uncovered environment in contrast to the closed environment provided by a coverslip and the substantial excess of reagents required for performing them in a Coplin jar. Assaying small samples with small and tightly controlled reagent volumes in a closed environment presents substantial complexity for instrument designers that, understandably, is preferably avoided. In these circumstances, evaporation that results from assay steps at high temperature becomes much more difficult to control than in a manual assay using excess reagents and covered sample slides.
Non-ionic detergents such as Nonidet P-40 or NP-40 have traditionally been used with reagents to facilitate spreading the reagents evenly over the surface of a sample slide. However, new slide designs utilize hydrophobic barriers painted on the slides to allow better control of the sample test area but at the same time make control of reagent flow properties more important. It has been found that detergents such as NP-40, which have been desirable because they enhance the flow properties of assay reagents, may disadvantageously cause reagents to spread outside the hydrophobic barrier of a sample slide resulting in loss of reagent making an automated assay more difficult to control.
Accordingly, there is a need to identify new reagents and methods for washing and aging biological samples in IHC, ISH and FISH assays. Such new reagents and methods would enhance assay procedures by enabling assays to be performed at high temperatures with minimal reagent evaporation without incurring sample degradation, allowing reagents to be applied in smaller and more tightly controlled volumes and enabling other problematic materials such as detergents to be eliminated from assay steps.